The present invention relates to a method for improving the growth and the detection of bacteria, yeasts, fungi, or cocci, by adding to the culture medium sterile-filtered yeast extract and/or p-iodonitrotetrazolium violet.
The method is especially suited for the detection of mycobacteria or germs under stress conditions, such as airborne germs after the stress of desiccation in the air.
Special embodiments of the method of the invention are described hereinbelow.
The growth of bacteria, yeasts, fungi, cocci and germs is performed commonly on culture media known for the purpose. Their detection can then be performed by colorimetric methods using appropriate indicators.
For bacteria, cocci and other germs, nutrient mediums are generally used, such as tryptic soy agar or broth; yeasts and fungi can be cultured, for example, on Sabouraud agar, broth or RMPI 1640 broth. Common methods for mycobacteria are performed on a basis of egg or egg jelly, such as Lxc3x6wenstein-Jensen or Stonebrinlk or agar media such as Middlebrook 7H10, 7H11, or fluid media such as Middlebrook 7H9 or Kirchner medium with 10% horse serum (DIN 58 943-3, Manual of Clinical Microbiology, 6th ed., 414-416).
For growth detection, the evaluation of C14 palmitic acid can be used, which releases 14CO2 (Bactec, Manual of Clinical Microbiology, 6th ed., 415); also oxygen consumption in the medium which is indicated by a fluorescent indicator (EP-A 0 509 781 Al, commercial name of the product MGIT) or by barometric measurement (ESP Automat fxc3xcr Blutkultur und Mykobalkterien, Difco) and by redox indicators such as resazurine/methylene blue (DE 4 316 394) or tetrazolium chloride (Deutsche Medizinische Wochenschrift 75, 1471 (1995)).
The growth of bacteria takes place relatively slowly. Mycobacteria on solid media require about 2-6 weeks, depending on the mycobacteria used for inoculation, and in liquid media 1-3 weeks. Growth detection with additional apparatus is very expensive, as is also the disposal of the radioactive waste. In known colorimetric redox indicators such as resazurine/methylene blue or tetrazolium chloride, the effect is seen that, for example, clinical strains of Mycobacteria tuberculosis are inhibited at low germ counts and do not grow satisfactorily except in high germ counts. However, even low germ counts have to be detected reliably in clinical test material. Furthermore, these indicators are toxic in the large amounts which are needed for low germ counts. Reducing the amount of these colorimetric redox indicators reduces the toxicity, but the result is that clinical isolates, such as those of Mycobacterium tuberculosis, do grow, but they are no longer colored, i.e., the desired colorimetric detection is not accomplished.
In the isolation and detection of airborne gents of various kinds, such as bacteria, cocci, yeasts, fungi and spores, the following problems occur: airborne germs are stressed by desiccation in the air. Moreover, the media for the detection of the germs are often gamma-sterilized, which again means stress for the medium and therefore leads to poorer growth of the germs to be detected. Both factors lead to the fact that germs of certain kinds, especially gram positive bacteria, and cocci, can no longer be reliably detected in the usual manner (colorimetry with resazurine).
The present invention is therefore addressed to the problem of developing a method by which the growth of bacteria, fungi, yeasts, cocci, can be improved and the detection of the targeted species can be performed reliably and at low cost.
This problem is solved according to the invention by adding to the culture medium sterile-filtered yeast extract and/or p-iodonitrotetrazolium violet.
It was found surprisingly that by the addition of sterile-filtered yeast extract or p-iodonitrotetrazolium violet the growth of bacteria, fungi, yeasts, cocci, can be accelerated. If the redox indicator p-iodonitrotetrazolium violet (INT) is added, the growth of the species under study can be detected simply by colorimetry, while the intensity of calorimetric detection with both additives is synergically increased by the addition of the sterile-filtered yeast extract.
The method of the invention is especially suited to the detection of mycobacteria, and especially clinical Mycobacterium tuberculosis isolates which are growing on various media such as 7H9 broth, 7H12, 7H9 with OADC ((oleic acid, albumin, dextrose, catalase, for Trademark, see Difco Manual) and PANTA (polymyxin, amphotericin B, nalidixic acid, trimethoprim, azlocillin) in the MGIT system Mycobacteria Growth Indicator Tube); in Kirchner medium with horse serum, and on solid media such as Lxc3x6wenstein Jensen.
Even bacteria, especially mycobacteria, damaged by long holding grow better with the additive according to the invention.
The time required for this purpose can be considerably reduced, and detection itself can be simplified.
For example, the sensitivity testing of mycobacteria, which otherwise is possible only within 1 week with the use of a radioactive substrate (14C palmitic acid in Becton Dickinson""s Bactex 460 system), can be evaluated even visually within 5 to 7 days by measuring turbidity upon the addition of sterile filtered yeast extract to 7H9 broth, without the need for high-cost systems such as radioactivity detection with the disposal problems which they entail. Furthermore, by the use of a combination with colorimetric substrates, namely INT, the color intensity of the indicator is increased.
The addition made according to the invention is also especially suited for the detection of air-borne germs, because the increase obtained in the germ count by sterile filtered yeast extract results in more rapid growth generally and especially in faster growth than in media that are not gamma-sterilized. Thus the calorimetric detection of germs, especially gram negative bacteria, yeasts and fungi, becomes possible if INT is added as indicator. This also permits the gamma-sterilization of media, and the sterile-filtered yeast extract compensates the damage to the media as regards growth properties. Thus, germs grow on gamma-sterilized media with sterile-filtered yeast extract even slightly better than on conventional, non-gamma sterilized media.
Tryptic soy agar or other such nutrient media can be used as the basic medium.
The addition of sterile-filtered yeast extract according to the invention is performed preferably in liquid form in amounts of 0.5 to 10 g/l, especially 0.5-5 g/l, and 2-2.5 g/l is very especially preferred.
The indicator, iodonitrotetrazolium violet (INT) is added to the culture medium preferably in an amount of 1 to 30 mg/l, especially 5-20 mg/l, and especially 8-15 mg/l.
Furthermore, the growth accelerated according to the invention can be detected calorimetrically in other ways, for example by now adding a small amount of the resazurine/methylene blue system (cf. DE 43 16 394), since the detection of even lower germ counts is possible due to the accelerated growth. This system can be varied in a known manner according to the purpose of the detection; for example, tuberculostatic agents can be added in the sensitivity testing of mycobacteria if growth control is desired. Also, redox stabilizers can be added in this case. The indicators are present in conventional amounts such as 1-200 mg/l and 5-100 mg/l of nutrient medium.
Growth can also be accelerated in other commercial systems by the addition of sterile-filtered yeast extract, as shown in Example 10 below, with Bactec 12B with sterile-filtered yeast extract in comparison with Example 11 (commercial Bactec 12B system), and in accordance with Examples 6, 7 and 8 in MGIT with sterile-filtered yeast extract, and sterile-filtered yeast extract plus INT and INT in comparison with Example 5 in commercial MGIT of Becton Dickinson.
With the addition made according to the invention, it is thus possible in general to accelerate the growth of bacteria, yeasts, cocci and fungi, and in addition to avoid the use of expensive detection apparatus.